The present invention relates, in general, to compositions and methods for the specific binding of a ligand to the stalk region of the polymeric immunoglobulin receptor for internalization into, or transport across, a cell.
One of the most challenging problems facing the pharmaceutical and biopharmaceutical industries is delivering therapeutic agents past the various semi-permeable membranes within the body. Particularly in the case of macromolecules, the obstacle to cost effective or convenient treatment is often due to the lack of an adequate drug delivery system. In turn, this issue dictates whether production of a drug is economically feasible. Thus the search for alternative delivery systems often rivals the search for new drugs themselves.
Gene transfer methods can be viewed as a paradigm of macromolecular drug delivery. These methods can be divided into three categories: physical (e.g., electroporation, direct gene transfer, and particle bombardment), chemical (e.g., proteinoids, microemulsions, and liposomes), and biological (e.g., virus-derived vectors, and receptor-mediated uptake). Amongst biological transfer methods, receptor-mediated uptake is a particularly promising approach. Targeting a ligand to an endocytosed receptor acts as a means to ferry that ligand into the cell. However, one drawback of receptor-mediated systems has been their general reliance on intravenous administration which severely limits their use.
Mucosal epithelial cells line a number of readily accessible tissues such as those found in the upper respiratory and gastrointestinal tracts. The accessibility of these cells make them an attractive target for drug delivery. See, e.g., Ferkol et al., J. Clin. Invest. 92:2394-2400 (1993); Ferkol et al., J. Clin. Invest. 95:493-502 (1995). Retrograde transport of an antibody from the lumenal to the basolateral surface of epithelial cells has been reported, albeit at very low levels. Breitfeld et al., J. Cell Biology 109:475-486 (1989). In that study, movement across the cell was followed by binding an antibody to the secretory component of polymeric immunoglobulin receptor (pIgR). Relative to the level of basolateral to apical transport, Breitfeld et al. reported that less than 5% of the transport was retrograde in nature. The nominal level of counter-transport minimizes the utility of secretory component as a means to deliver biologically active compositions into cells. Moreover, due to the abundance of cleaved pigR in the lumen, binding of ligand to cleaved pIgR, rather than the intact pigR of the cell surface, would diminish the utility of pIgR counter-transport as a mechanism of drug delivery.
Accordingly, what is needed in the art is a means to convey ligands into or across a cell surface with high efficiency. More particularly, what is needed in the art is a means to deliver macromolecules to, into, or across cells lining the gastrointestinal or respiratory tracts. The present invention provides these and other advantages.
In one aspect, the present invention is directed to a ligand that binds specifically to the stalk of a polymeric immunoglobulin receptor (pIgR) of a cell with the proviso that the ligand does not substantially bind to secretory component of pIgR under physiological conditions. Typically, the antibody specifically binds only to the stalk. In one embodiment, the ligand is an antibody, preferably a humanized antibody. In another embodiment the ligand is a recombinant single chain variable region fragment of an antibody. In yet another embodiment the ligand binds to an extracellular epitope within the first 33 amino acids that are cell membrane proximal to a cleavage site of the receptor.
The ligand may comprise a binding component for binding to the stalk, and a biologically active component such as a nucleic acid, protein, radioisotope, lipid, and carbohydrate. Biologically active components comprise anti-inflammatories, anti-infectives, anti-sense oligonucleotides, antibiotics, and anti-infectives. In one embodiment, the biologically active component is a nucleic acid encoding a wildtype cystic fibrosis transmembrane conductance regulator. In a preferred embodiment the cell is an epithelial cell, most preferably a mammalian epithelial cell.
In another aspect, the present invention is directed to a method of introducing a ligand into a cell expressing a polymeric immunoglobulin receptor by attaching the ligand to the stalk of the polymeric immunoglobulin receptor of the cell with the proviso that the ligand does not substantially bind to the secretory component of pIgR under physiological conditions. In one embodiment, the ligand is attached to the stalk at the apical surface of a cell; and in further embodiments, the ligand is transcytosed to the basolateral surface of the cell, and released from the stalk at the basolateral surface.
In a further aspect, the present invention relates to a method of attaching a ligand to a cell expressing a polymeric immunoglobulin receptor comprising the step of binding the ligand to a stalk of the receptor with the proviso that the ligand does not substantially bind to secretory component of pIgR under physiological conditions. In one embodiment, the ligand is introduced into the cell after binding. Alternative embodiments of the present invention may be had by reference to the various aspects of the present invention.
Amongst the various in vivo and in vitro utilities, the present invention may be used to transport therapeutic or diagnostic compositions to, into, or across, mucosal epithelial cells. Thus, the invention provides a highly efficient and convenient means to transfer nucleic acids or proteins into epithelial cells.
The present invention is directed to a ligand that binds specifically to the stalk of a polymeric immunoglobulin receptor (pIgR) of a cell with the proviso that the ligand does not substantially bind to secretory component of pIgR under physiological conditions. The invention provides, inter alia, methods of attaching and introducing a ligand into a cell expressing pIgR.
After transport to the apical surface of epithelial cells, the majority of pIgR is cleaved and secretory component is released. We have discovered that, surprisingly and unexpectedly, cleavage of intact pIgR in the lumen leaves a residual extracellular region of pIgR (i.e., the xe2x80x9cstalkxe2x80x9d) intact; and further, the stalk remains accessible to binding despite the abundance of proteases typically present in lumenal milieu. The ability to bind, endocytose, and transcytose a ligand bound to the pIgR stalk provides, in part, the invention as disclosed and claimed herein.
The present invention has utility as a means of transporting therapeutic or diagnostic compositions to, into (endocytosis) or across (transcytosis) a cell expressing pIgR. Thus the invention can be used to transport biologically active compositions such as proteins, nucleic acids, or detectable labels specifically to cells expressing pIgR. The invention also provides a means of labeling and distinguishing epithelial cells from amongst a mixed cell population in pathological studies. Further, since pIgR expression is reduced in carcinomas relative to normal epithelium, the labeling of pIgR has utility as a diagnostic adjunct in endoscopic or radiologic procedures. Additionally, binding of therapeutic ligands to plgr has utility in extending their duration in the lumen of various passageways and increasing their effectiveness.
Definitions
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, second edition, John Wiley and Sons (New York), and Hale and Marham (1991) The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. provide one of skill with a general dictionary of many of the terms used in this invention. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. For purposes of the present invention, the following terms are defined below.
By xe2x80x9cligandxe2x80x9d or xe2x80x9cligand binding moietyxe2x80x9d, is meant all molecules capable of specifically binding to the polymeric immunoglobulin receptor (pIgR). Ligands include, but are not limited to, antibodies, proteins, peptides, nucleic acids, lipids, and carbohydrates.
By xe2x80x9cbiologically active componentxe2x80x9d is meant a compound which, in vivo, directly causes or inhibits an increase or decrease in cellular transcription, translation, receptor binding, active or passive transport, cell signaling, signal transduction, cell division, cell differentiation, cell death, cell adhesion, cell movement, cell morphology, metabolism, enzyme activity, apoptosis, protein degradation, protein movement (e.g., secretion), protein stability, or phosphorylation. Biologically active components also comprise diagnostic compositions which allow the foregoing events to be assessed.
By xe2x80x9cbind(s) specificallyxe2x80x9d or xe2x80x9cspecifically bind(s)xe2x80x9d or xe2x80x9cattachedxe2x80x9d or xe2x80x9cattachingxe2x80x9d is meant the preferential association of a ligand, in whole or part, with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, specific binding, may be distinguished as mediated through specific recognition of the target molecule. Typically specific binding results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the bound molecule and cells lacking the target molecule. Specific binding typically results in greater than 2 fold, preferably greater than 5 fold, more preferably greater than 10 fold and most preferably greater than 100 fold increase in amount of bound ligand (per unit time) to a cell or tissue bearing the target molecule as compared to a cell or tissue lacking the target molecule or marker.
By xe2x80x9cstalkxe2x80x9d is meant the extracellular component of the polymeric immunoglobulin receptor (pIgR) that corresponds to that region of pIgR that is bound to the cell following cleavage of that segment of pIgR which constitutes the secretory component. The stalk is present regardless of whether the segment of pIgR which corresponds to secretory component is cleaved or uncleaved from pIgR.
By xe2x80x9cpIgRxe2x80x9d or xe2x80x9cpolymeric immunoglobulin receptorxe2x80x9d is meant the receptor which is expressed in mucosal epithelial cells, including airway epithelial cells, submucosal gland cells, intestinal cells, nasal epithelium, breast, oral mucosa, urinary and reproductive tract epithelium, and conjunctival tissue, and is implicated in basolateral to apical transcytosis of dimeric immunoglobulin A (digA) and/or pentameric IgM.
By xe2x80x9cnot substantially bindxe2x80x9d is meant that no more than 15% of a ligand which specifically binds to a target molecule is bound to a particular non-target molecule. More preferably, no more than 10% is bound to the non-target molecule, even more preferably less than 5%, and most preferably less than 1%.
By xe2x80x9csecretory componentxe2x80x9d is meant that extracellular portion of pIgR which is generally cleaved following basolateral to apical transcytosis. Typically, the secretory component comprises the dimeric IgA (dIgA) binding portion of pIgR. Secretory component is typically released into the lumen with or without dIgA bound to the secretory component.
By xe2x80x9cphysiological conditionsxe2x80x9d is meant an extracellular milieu having conditions (e.g., temperature, pH, and osmolarity) which allows for the sustenance or growth of a cell of interest.
By xe2x80x9cantibodyxe2x80x9d is meant an immunoglobulin molecule obtained by in vitro or in vivo generation of the humoral response, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and recombinant single chain Fv fragments (scFv). The term xe2x80x9cantibodyxe2x80x9d also includes antigen binding forms of antibodies (e.g., Fab, F(ab)2).
By xe2x80x9chumanized antibodyxe2x80x9d is meant an antibody which comprises a non-human amino acid sequence but whose constant region has been altered to reduce immunogenicity in humans.
By xe2x80x9cwildtype cystic fibrosis transmembrane conductance regulatorxe2x80x9d is meant a functional form of the cystic fibrosis transmembrane conductance regulator (CFTR). Riordan et al., Science, 245:1066-1073 (1989).
By xe2x80x9capical surfacexe2x80x9d is meant that surface of a cell to which intact pIgR is transcytosed to after endocytosis from the basolateral surface. Generally, the apical surface of the cell adjoins a lumen and therein intact pIgR is cleaved to release the secretory component.
By xe2x80x9cbasolateral surfacexe2x80x9d is meant that surface of a cell from which intact pIgR is delivered to after synthesis in the endoplasmic reticulum and passage through the Golgi complex.
By xe2x80x9csurface of the stalkxe2x80x9d is meant the extracellular region of the stalk.
By xe2x80x9creleasedxe2x80x9d is meant the interference in the specific association of a ligand, in whole or part, with its target molecule.
By xe2x80x9ctranscytosedxe2x80x9d or xe2x80x9ctranscytosisxe2x80x9d is meant conveyance from one plasma membrane of the cell to another via an intracellular route. Typically, transcytosis occurs from the basolateral to apical or apical to basolateral plasma membrane of the cell.
By xe2x80x9ccell membrane proximalxe2x80x9d is meant next to or nearer the cell membrane.
By xe2x80x9cextracellularxe2x80x9d is meant the region extending outward from the lipid bilayer encompassing a cell.
Identification of pIgR
The nucleic acid and amino acid sequence of the polymeric immunoglobulin receptor has been identified in a variety of taxonomically diverse species. See, Piskurich et al., Journal of Immunology 154:1 735-1747 (1995). Identification of pIgR from other species can be accomplished by any number of methods well known to those of skill in the art. For example, using published pIgR sequences a nucleic acid probe to plgR can be constructed. The probe typically should be derived from a conserved region of plgR. Hybridization of the probe to a genomic or cDNA library can be used to identify pIgR in an unknown species. It will be understood by the skilled artisan that the nucleic acid sequence of the pIgR probe should generally be that of the species most closely related to the probed species. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Acid Probes Part I, Chapter 2 xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid probe assaysxe2x80x9d, Elsevier, N.Y.
In an alternative approach, pIgR or peptide fragments thereof (e.g., secretory component) can be used to create antibodies to screen expression libraries. See, e.g., Ferkol et al., J. Clin. Invest. 95:493-502 (1995). These and other methods well known to the skilled artisan may be found, for example, in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, lnc., San Diego, Calif. (Berger); Sambrook et al. (1989) Molecular Cloningxe2x80x94A Laboratory Manual (2nd ed.) Vol. 1-3; and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1994 Supplement) (Ausubel). Confirmation of the identity of a nucleic acid or protein as encoding pIgR may be had by such approaches as constructing antibodies to the putative pIgR protein and confirming the ability of these antibodies to bind to a protein having the characteristics of pIgR (e.g., being present on the surface of epithelial cells, binding of dimeric IgA or pentameric IgM, etc.).
Identification of the Stalk
The stalk can be identified by a variety of techniques well known to those of skill. A putative heptapeptide consensus sequence which identifies the cleavage site of pIgR and thereby defines the amino terminus of the stalk has been identified. The sequence Phe-Ala-Xaa-Glu (SEQ ID NO:1), where Xaa is a polar or charged amino acid, was identified as immediately preceding this putative cleavage site. Piskurich et al., Journal of Immunology 154:1735-1747 (1995). Cleavage at the consensus site liberates the secretory component and defines its carboxy terminus. The carboxy terminus of secretory component may be altered by secondary cleavage events (e.g., exopeptidase or endopeptidase activity) to yield secondary carboxy termini. Id. However, the amide linkage which initially defines the amino terminus of the stalk and the carboxy terminus of secretory component may be identified by sequence alignment and identification of the cleavage consensus sequence.
Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482; by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443; by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA); the CLUSTAL program is well described by Higgins and Sharp (1988) Gene, 73: 237-244 and Higgins and Sharp (1989) CABIOS 5: 151-153; Corpet, et al. (1988) Nucleic Acids Research 16, 10881-90; Huang, et al. 1992) Computer Applications in the Biosciences 8, 155-65, and Pearson, et aL (1994) Methods in Molecular Biology 24, 307-31. Alignment is also often performed by inspection and manual alignment.
In another approach, secretory component can isolated from fluids in the apical lumen (e.g., milk or bile) and sequenced by amino acid sequencing methods well known to those of skill such as Edman degradation, or mass spectrometry. Eiffert et al., Hoppe-Seyler""s Z. Physiol. Chem. 365:1489-1495 (1984). Amongst the various secondary carboxyl ends defined by secondary cleavage events, the carboxy terminal amino acid adjacent to the cleavage site can be identified.
Peptides which correspond to the pIgR stalk of selected species include:
Mouse: Glu-Arg-Glu-Ile-Gln-Asn-Ala-Gly-Asp-Gln-Ala-Gln-Glu-Asn-Arg-Ala-Ser-Gly-Asn-Ala-Gly-Ser-Ala Gly-Gly-Gln-Ser-Gly-Ser-Ser-Lys(SEQ ID NO:2)
Rat: Glu-Arg-Glu-lIe-Gln-Asn-Ala-Gly-Asp-Gln-Ala-Gln-Glu-Asn-Arg-Ala-Ser-Gly-Asn-Ala-Gly-Ser-Ala-Gly-Gly-Gln-Ser-Gly-Ser-Ser-Lys(SEQ ID NO:3)
Human: Glu-Lys-Ala-Val-Ala-Asp-Thr-Arg-Asp-Gln-Ala-Asp-Gly-Ser-Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-GIn-Gly-Gly-Ser-Ser-Arg (SEQ ID NO:4)
Bovine: Glu-Ser-Val-Lys-Asp-Ala-Ala-Gly-Gly-Pro-Gly-Ala-Pro-Ala-Asp-Pro-Gly-Arg-Pro-Thr-Gly-Tyr-Ser-Gly-Ser-Ser-Lys(SEQ ID NO:5)
Rabbit: Leu-Ala-Glu-Val-Ala-Val-GIn-Ser-Ala-Glu-Asp-Pro-Ala-Ser-Gly-Asp-Pro-Ala-Ser-Gly-Ser Arg-Ala-Ser-Val-Asp-Ser-Gly-Ser-Ser-Glu-Glu-Gln Gly-Gly-Ser-Ser-Arg-Ser-Lys (SEQ ID NO:6)
Cells Expressing pIgR
While the present invention broadly pertains to eukaryotic cells. The pIgR expressing cell of the present invention is preferably a mammalian cell and more preferably a mammalian epithelial cell that normally secretes IgA. Mammalian cells can be transfected with a nucleic acid encoding pIgR isolated, synthesized or otherwise derived from one or more desired species. Methods of transfecting and expressing genes in mammalian cells are known in the art. Transducing cells with viral vectors can involve, for example, incubating viruses with cells within the viral host range under conditions and concentrations necessary to cause infection. See, e.g., Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego, Calif.(D.V. Goeddel, ed.) (1990) or M. Krieger, Gene Transfer and Expressionxe2x80x94A Laboratory Manual, Stockton Press, New York, N.Y., (1990) and the references cited therein.
The culture of cells which can be used in the present invention include cell lines and cultured cells from tissue or blood samples is well known in the art. Freshney (Culture of Animal Cells, a Manual of Basic Technique, third edition Wiley-Liss, N.Y. (1994)) and the references cited therein provides a general guide to the culture of cells. The nucleic acid sequences encoding pIgR from the desired species may be expressed in a variety of eukaryotic host cells, including yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines as well as MDCK and human colon carcinoma derived cells such as Caco2. The recombinant protein gene will be operably linked to appropriate expression control sequences for each host. For eukaryotic cells, the control sequences will include a promoter and preferably an enhancer derived, for example, from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
Binding Ligands to the Stalk
The specific stalk binding ligand is not critical to this invention and various ligands may be used. A host of methods for construction and selection of ligands such as nucleic acids, proteins or peptides (collectively, xe2x80x9cpeptides), or antibodies, or small organics or inorganics (e.g., U.S. Pat. No. 5,143,854; WO 90/15070; WO 92/10092; WO 96/11878) having the desired specific binding characteristics are well known in the art. Preferably, ligands of the present invention will, under physiological conditions, bind to the stalk without substantially binding to the secretory component of pIgR. More preferably, the ligands of the present invention specifically bind only to the stalk under physiological conditions. Typical physiological conditions vary from tissue to tissue. However, exemplary physiological conditions are encountered in the gastrointestinal or respiratory tract of mammals, including but not limited to humans. A ligand may be chosen to bind to an extracellular ligand binding site (xe2x80x9cepitopexe2x80x9d) contained within the first 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33 membrane proximal amino acids of the stalk.
Antibodies, including polyclonal, monoclonal, or recombinant single chain Fv antibodies, can be constructed for use as ligands in the present invention. Methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, N.Y.; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature 256: 495-497; See, Huse et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. Birch and Lennox, Monoclonal Antibodies: Principles and Applications, Wiley-Liss, N.Y., N.Y. (1995).
Other suitable techniques for antibody or peptide ligand preparation include selection of libraries of recombinant antibodies/peptides in phage or similar vectors. High affinity antibodies and peptides to the stalk can be rapidly isolated by using phage display methods to express recombinant single chain Fv (scFv) fragments or peptide ligands on the phage surface. Briefly, genes encoding the surface protein of a phage are altered so as to allow the insertion of an antibody or peptide gene which is expressed as a fusion protein on the surface of the phage that carries the gene. The phage expressing the desired antibody or peptide ligand can be selectively enriched and isolated by virtue of its affinity/avidity for the stalk. The DNA encoding the ligand is packaged in the same phage and which allows the gene encoding the ligand to be isolated. A variety of such methods are amply discussed in the literature and well known to the skilled artisan. See, e.g., Winter et aL, Annu. Rev. Immunol. 12:433-455 (1994); Marks et al., J. MoL Biol. 222:581-597 (1991); Vaughan et al., Nature Biotechnology 14:309-314 (1996), U.S. Pat. Nos. 4,642,334; 4,816,397; 4,816,567; 4,704,692; WO 86/01533; WO 88/09344; WO 89/00999; WO 90/02809; WO 90/04036; EP 0 324 162; EP 0 239 400.
In chemical peptide synthesis, a procedure termed xe2x80x9cDivide, Couple and Recombinexe2x80x9d (DCR) has been used to produce combinatorial peptide libraries. See, Furka et al, Int. J. Pept. Protein Res. 37:487-493 (1991) and Houghten et al., Nature 354:84-86 (1991). As an alternative to DCR, peptide mixtures have also been made by direct coupling of monomer mixtures. See, Rutter et al., U.S. Pat. No. 5,010,175. The use of such methods to produce mixtures of other linear polymers, such as xe2x80x9cpeptoidsxe2x80x9d, has been suggested. See, Simon, et al, Proc. Natl. Acad. Sci. USA 89:9367-9371 (1992). In oligonucleotide synthesis, xe2x80x9cdegeneratexe2x80x9d or xe2x80x9cwobblexe2x80x9d mixtures of oligonucleotide products can be made by, for example, delivery of equimolar mixtures of monomers to an oligonucleotide polymer at specific steps during synthesis. See, Atkinson and Smith, in xe2x80x9cOligonucleotide Synthesis. A Practical Approachxe2x80x9d, 1984, IRL Press, Oxford, edited by M. Gait, pp 35-81. These methods of synthesizing peptides or oligonucleotides provide large numbers of compounds for testing which, if active, can be readily identified.
Preferably, ligands will be constructed to minimize immunogenicity in the host as, for example, by maximizing the number of autologous (self) sequences present in the ligand. Accordingly, chimeric antibodies having non-xenogenic variable regions are preferred. Particularly preferred are the use of antibodies in which xenogenic portions are excluded, or are essentially limited to the complementarity determining regions as in humanized antibodies.
Ligand Binding and Testing
Binding (i.e., attachment) of the ligand to the stalk of pIgR may occur prior or subsequent to cleavage of secretory component; and the ligand may be attached at the basolateral or apical surface. Thus, the ligand can be endocytosed basolaterally or apically, or be subject to apical to basolateral, or basolateral to apical transcytosis. The fate of the ligand, or any element thereof, will vary according to its physico-chemical characteristics. Accordingly, the properties of the ligand may be selected or designed to perform the desired function at the cell surface, within the endosome, or following transcytosis. For example, varying the sensitivity of a ligand to proteolytic or reducing environments can be used to determine the distribution of ligand bound, internalized, or transported across the cell. Where desirable, a ligand may be designed to remain specifically bound to the cell following attachment or transcytosis or, alternatively, to be released into the extracellular milieu. Thus, the properties of any of the various elements of the ligand, including the binding component, biologically active component or linker, may be designed or selected to allow for different degrees of affinity, stability, or activity at different intracellular compartments or surfaces of the cell, as desired.
A. Ex Vivo Testing of Ligand Binding
In vitro binding of the ligand to the stalk may be conveniently assessed by measuring endocytosis or transcytosis of bound ligand in mammalian epithelial cells. xe2x80x9cEndocytosisxe2x80x9d refers generally to the phenomenon of a cell ingesting material, e.g., by phagocytosis or pinocytosis. Receptor-mediated endocytosis provides an efficient means of causing a cell to ingest material which binds to a cell surface receptor. See, Wu and Wu (1987) J. Biol. Chem. 262:4429-4432; Wagner et al. (1990) Proc. Natl. Acad. Sci. USA 87:3410-3414, and EP-A1 0388 758. Any number of well known methods for assaying endocytosis may be sued to assess binding. For example, binding, transcytosis, and internalization assays are described at length in Breiftfeld et al. J. Cell Biol. 109:475-486 (1989).
Apical endocytosis is conveniently measured by binding a ligand such as a Fab fragment to the stalk at the apical surface of Madin-Darby canine kidney (MDCK) cells at 4xc2x0 C., warming to 37xc2x0 C. for brief periods (0-10 min), and cooling the cells back down to 4xc2x0 C. Methods of pIgR expression in MDCK cells is well known in the art. Breitfeld et al., Methods in Cell Biology 32:329-337 (1989). Fab remaining on the surface are removed by stripping at pH 2.3. Intracellular Fab are those that remain cell-associated after the stripping, while surface-bound Fab are those removed by the acid wash. Controls for non-specific sticking include using pre-immune Fab and/or MDCK cells that are not transfected with pIgR.
Transcytosis can be readily assessed by allowing MDCK cells to bind the Fab at the apical surface at 4xc2x0 C., warming up to 37xc2x0 C. for 0-240 min, and then measuring the amount of Fab delivered into the basolateral medium. This basolaterally-delivered Fab is compared to the sum of Fab that remains associated with the cells (intracellular or acid-stripped) and the Fab released back into the apical medium. Alternatively, transcytosis can be assessed by continuously exposing cells to the Fab in the apical medium and measuring accumulation of Fab in the basolateral medium. This method avoids cooling the cells, but does not provide the kinetics of transporting a single cohort of ligand. In both methods degradation of the Fab can be assessed by running aliquots of the transcytosed Fab on SDS-PAGE and probing a Western with anti-chicken antibodies. Non-specific transport (e.g. due to fluid phase endocytosis and transcytosis, or paracellular leakage between cells) can be controlled for by using MDCK cells that are not transfected with the pIgR and/or pre-immune Fab.
B. In Vivo Testing of Ligand Binding
Transcytosis in vivo may conveniently be assessed using pathogen-free experimental animals such as Sprague-Dawley rats. Labelled ligand (e.g., radioiodinated antibody) can be administered into the nares. As will be understood by those of skill in the art, a xe2x80x9clabelxe2x80x9d is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means such as fluorescence, chemifluoresence, or chemiluminescence. Apical to basolateral transcytosis can be readily determined by measuring delivery of the ligand into the circulation as determined by the presence of label. The integrity of the ligand recovered from the circulation can be assessed by analyzing the ligand on SDS polyacrylamide gel electrophoresis.
Biologicallv Active Component
The biologically active component of the ligand may be covalently or non-covalently bound to the ligand. For example, chelators may be used to bind various isotopes, or nucleic acids may be bound to the ligand via hydrogen bonds. Biologically active components may also be encompassed within emulsions, proteinoids, or liposomes. As those skilled in the art will understand, such structures may be linked covalently to the ligand via derivatized polar head groups or via membrane integral proteins. Binding components of the ligand may also comprise the biologically active component.
Biologically active components comprise any number of compounds known to those of skill as anti-inflammatories, cytokines, anti-infectives, enzyme activators or inhibitors, allosteric modifiers, or antibiotics. Thus, biologically active component includes such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. The term xe2x80x9cnucleic acidxe2x80x9d refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides. Nucleic acids include anti-sense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, triplex forming oligonucleotides, or nucleic acids encoding proteins or peptides. Nucleic acids also includes compositions for gene therapy such as those encoding for the wildtype cystic fibrosis transmembrane conductance regulator.
Attaching Biologically Active Compositions to the Ligand
The procedure for attaching a biologically active component to a ligand will vary according to the chemical structure of the component. Generally, the ligands will contain a variety of functional groups which are available for reaction with a suitable functional group on a biologically active molecule to bind the agent thereto. Alternatively, the ligand and/or biologically active component may be derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Ill. A xe2x80x9clinkerxe2x80x9d as used herein refers to a molecule used to join, covalently or non-covalently, the ligand and biologically active component. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. See, e.g., Birch and Lennox, Monoclonal Antibodies: Principles and Applications, Chapter 4, Wiley-Liss, New York, N.Y.(1995); U.S. Pat. Nos. 5,218,112, 5,090,914; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif.(1996).
Where both molecules are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine). A bifunctional linker having one functional group reactive with a group on a particular biologically active component, and another group reactive with a ligand, may be used to form the desired conjugate. Alternatively, derivatization may involve chemical treatment of the component; e.g., glycol cleavage of the sugar moiety of the glycoprotein antibody with periodate to generate free aldehyde groups. The free aldehyde groups on the antibody may be reacted with free amine or hydrazine groups on an agent to bind the agent thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation of free sulfhydryl groups on antibodies or antibody fragments are also known (See U.S. Pat. No. 4,659,839). Many procedure and linker molecules for attachment of various compounds including radionuclide metal chelates, toxins and drugs to proteins such as antibodies are known. See, for example, European Patent Application No. 188,256; U.S. Pat. Nos.,4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987)).
It is sometimes desirable to release the conjugated molecule when it has reached a target site. Therefore, conjugates comprising linkages which are cleavable in the vicinity of the target site may be used. Cleaving of the linkage to release the biologically active component from the antibody may be prompted by enzymatic activity or conditions to which the conjugate is subjected either inside the target cell or in the vicinity of the target site. When the target site is a tumor, a linker which is cleavable under conditions present at the tumor site (e.g. when exposed to tumor-associated enzymes or acidic pH) may be used. Use of the cis-aconitic acid spacer is useful for releasing biologically active components in endosomes. Similarly, disulfide linkages are cleavable in the reductive environment of the endosomes.
A number of different cleavable linkers are known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. The mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis. U.S. Pat. No. 5,141,648 discloses immunoconjugates comprising linkers of specified chemical structure, wherein the linkage is cleaved in vivo thereby releasing the attached compound (radiotherapeutic agent, drug, toxin, etc.). The linker is susceptible to cleavage at a mildly acidic pH, and is believed to be cleaved during transport into the cytoplasm of a target cell, thereby releasing the biologically active compound inside a target cell. U.S. Pat. No. 4,671,958 includes a description of immunoconjugates comprising linkers which are cleaved at the target site in vivo by the proteolytic enzymes of the patient""s complement system. In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other components to ligands one skilled in the art will be able to determine a suitable method for attaching a given component to a ligand of the present invention.
Egress of the Ligand from the Endosome
A number of methods well known to the skilled artisan may be used to transport ligand, or any portion thereof, out of the endosome.
A poly-L-lysine/nucleic acid complex bound to a ligand which binds specifically to the stalk can be used for efficient transfection. Ferkol et al., J. Clin. Invest., 92:2394-2400 (1993); and Ferkol et al., J. Clin. Invest., 95:493-502 (1995).
In another approach, poly-L-lysine can be linked, such as by genetic fusion or chemical linkers, to a ligand that binds specifically to the stalk of pIgR. In turn, this complex can be linked to defective adenovirus. Curiel and co-workers have demonstrated that naked plasmid DNA bound electrostatically to poly-L-lysine or poly-L-lysine-transferrin which has been linked to defective adenovirus mutants can be delivered to cells with transfection efficiencies approaching 90%. The adenovirus-poly-L-lysine-DNA conjugate binds to the normal adenovirus receptor and is subsequently internalized by receptor-mediated endocytosis. This approach has been used to obtain as much as a 1000-fold increase in expression of gene therapy vectors. Herpes viruses have similar properties. Curiel et al (1991) Proc Natl Acad Sci USA 88:8850-8854; Cotten et al (1992) Proc Natl Acad Sci USA 89:6094-6098; Curiel et al (1992) Hum Gene Ther 3:147-154; Wagner et al. (1992) Proc Natl Aced Sci USA 89:6099-6103; Michael et al. (1993) J Biol Chem 268:6866-6869; Curiel et al (1992) Am J Respir Cell Mol Biol 6:247-252, and Harris et al. (1993) Am J Respir Cell Mol Biol 9:441-447); Gao et al. (1993) Hum. Gene Ther. 4:17-24; Curiel et al U.S. patent application Ser. No. 07/768,039.
In yet another approach using influenza virus, a hydrophobic peptide in the hemagglutinin can act as a fusion peptide at low pH to effect fusion of the virus with the membrane of the endosome and delivering the virus into the cytoplasm. This peptide has been used in transferrin/peptide/poly-L-lysine/DNA complexes for gene transfer using the transferrin receptor and substantially improved the efficiency of expression. Wagner et al., Proc. Natl. Acad. Sci USA 89:7934-7938 (1992). This peptide can be engineered into a ligand for transport of the ligand, or a portion thereof, out of the endosome.
A further approach may employ ricin A. Ricin A chain is capable of penetrating out of endosome and into the cytosol. Beaumell et al., J. Biol. Chem. 268:23661-23669 (1993). A ligand of the present invention may be linked to ricin A, such as by genetic fusion or chemical linkers.
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.